The goal of the proposed research is to identify the molecular interactions among the proteins which constitute the skeletal muscle calcium release unit (CRU) and which have been shown to be functionally essential for normal excitation-contraction (e-c) coupling: RyR1, DHPR, triadin and junctin. It is the central tenant of this project and program that the most appropriate method that can be used to study the function of CRU proteins is to first remove them and then replace them in the context of a muscle cell. This method also makes it possible to mutate these CRU proteins thus facilitating the examination of critical protein-protein interactions. In Project 1 we will create key reagents to allow study of the mechanisms of e-c coupling in genetically engineered muscle cells and will study the physiology of their Ca 2+ movements. The mdg mouse is naturally deficient in alpha1s-DHPR expression, we have engineered "knock out" mice that are deficient in RyR1 expression and we have been given mice that are deficient in Homer to study their skeletal muscle. We and the other members of the program have and will continue to use these mice to facilitate our studies of how the als-DHPR and RyR1 interact to engage skeletal e-c coupling and to provide critical information on the roles that calmodulin and homer may play on this interaction and/or on the control of Ca 2+ release in the muscle cell. We propose here to create mice deficient in two other key triad proteins, triadin and junctin, to determine their role in e-c coupling and "knock in" mice expressing mutated alpha1s-DHPRs to determine the role of Ca 2+ current on skeletal e-c coupling in vivo. We believe that these models will provide the basis for major advances in unlocking the mechanisms of CRU signaling essential for normal e-c coupling. A crucial extension of our work is to define the molecular structure of RyR1 in its lipid environment. Direct 2D electron crystal analysis of RyR1 at a resolution of 3-10 angstrom will allow us for the first time to understand the molecular structural basis of functional modifications of RyR1.